Establishment of adventitious root co-culture of Ginseng and Echinacea for the production of secondary metabolites

We have successfully established the co-culture of ginseng (Panax ginseng C.A. Meyer) and echinacea [Echiancea purpurea (L.) Moench.] adventitious roots for the production secondary metabolites. Adventitious roots of ginseng and echinacea were cultured in different proportions (5 g L⁻¹; 4:1, 3:2 and 2:1 ginseng and echinacea, respectively) in 5-L capacity airlift bioreactors containing 4 L Murashige and Skoog medium supplemented with 25 μM indole-3-butyric acid and 50 g sucrose L⁻¹ and maintained at 25°C in the dark for 40 days. Results showed the negative effect of echinacea adventitious roots on the growth of ginseng roots, however, by limiting the inoculum density of echinacea, it was possible to establish the co-cultures. To enhance the accumulation of secondary metabolites, co-cultures were treated with 200 μM methyl jasmonate after 30 days of culture initiation. Methyl jasmonate elicitation promoted the accumulation of ginsenosides in the co-cultures. It was possible to produce ginsenosides and caffeic acid derivatives in higher amounts by establishing co-cultures with higher inoculum proportion of ginseng to echinacea (4:1 and 3:2) followed by elicitation treatment. This work demonstrates the effectiveness of interspecies adventitious root co-cultures for the production of plant secondary metabolites.     

Overexpression of a chrysanthemum transcription factor gene <Emphasis Type="Italic">DgNAC1</Emphasis> improves drought tolerance in chrysanthemum

Phenolic acids and tanshinones are two groups of pharmaceutical components present in Salvia miltiorrhiza Bunge. Methyl jasmonate (MeJA) has been reported to influence the accumulation of both phenolic acids and tanshinones in S. miltiorrhiza hairy roots. However, there is currently a lack of information regarding the comparison of how these two groups of bioactive compounds in S. miltiorrhiza respond to MeJA under the same conditions. In the present study, the effect of 100 µM MeJA on the biosynthesis of phenolic acids and tanshinones in S. miltiorrhiza hairy roots was investigated. The results showed that MeJA dramatically induced the accumulation of five different phenolic acids, especially rosmarinic acid and salvianolic acid B, which reached their highest contents at day 3 (20.3 mg/g DW, 1.5-fold of control) and day 6 (47.49 mg/g DW, 2.5-fold of control), respectively. The total production of phenolic acids was induced by as much as 3.3-fold of the control (day 9 after treatment), reaching 357.5 mg/L at day 6. However, tanshinone I was almost unaffected by MeJA treatment, and the accumulation of tanshinone IIA was inhibited. Furthermore, cryptotanshinone and dihydrotanshinone I were moderately induced by MeJA. The gene expression results indicated that MeJA probably induced the whole pathways, especially the tyrosine-derived pathway and the methylerythritol phosphate pathway, and finally resulted in the increased production of these metabolites. This study will help us to further understand how the different biosynthetic mechanisms of phenolic acids and tanshinones respond to MeJA and provide a reference for the future selection of elicitors for application to improving the production of targeted compounds.     

Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags

Expressed sequence tags (ESTs) provide a valuable tool that can be used to identify genes in secondary metabolite biosynthesis. Ginseng (Panax ginseng C.A Meyer) is a medicinal plant that accumulates ginsenosides in roots. We sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. ESTs involved in ginsenoside biosynthesis were identified by a keyword search of BLASTX results and a domain search of ginseng ESTs. We identified 4 oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, 9 nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone.Abbreviations cDNA Complementary DNA - ESTs Expressed sequence tagsCommunicated by I.S. Chung     

雷公藤AP2/ERF转录因子基因的克隆与分析

该研究以雷公藤发状根为材料,根据雷公藤根转录组数据设计引物,采用RT-PCR方法克隆得到2个雷公藤AP2/ERF转录因子,分别命名为TwAP2/ERF1基因(GenBank登录号:GAVZ01042389.1)和TwAP2/ERF2基因(GenBank登录号:GAVZ01016765.1)。TwAP2/ERF1基因含有一个525bp开放阅读框(ORF),编码186个氨基酸;TwAP2/ERF2基因的ORF为789bp,编码262个氨基酸;2个基因编码的蛋白质均为亲水性蛋白质。系统进化分析表明,TwAP2/ERF1与油桐(Vernicia fordii)AP2/ERF(APQ47444.1)和木油桐(Vernicia montana)AP2/ERF(APQ47365.1)相似性较高,TwAP2/ERF2与毛果杨(Populus trichocarpa)AP2/ERF(XP_002304640.1)和樱桃(Prunus pseudocerasus)AP2/ERF(ALD84477.1)相似性较高。雷公藤发状根经MeJA诱导后,TwAP2/ERF1基因的相对表达量明显提高,并于处理后9h达到最高值,为对照表达量的16.77倍;而MeJA处理对TwAP2/ERF2基因的表达表现出抑制作用,但于处理后48h相对表达量有所提高。研究表明,雷公藤TwAP2/ERF1转录因子响应MeJA早期诱导正调控,推测其可能参与调控雷公藤植物次生代谢产物的生物合成,该研究结果为阐明雷公藤次生代谢物质的生物合成调控与利用现代生物技术提高雷公藤植物细胞中次生代谢物质的含量奠定了基础。     

Integrative transcriptome analysis identifies new oxidosqualene cyclase genes involved in ginsenoside biosynthesis in Jilin ginseng

BackgroundJilin ginseng, Panax ginseng, is a valuable medicinal herb whose ginsenosides are its major bioactive components. The ginseng oxidosqualene cyclase (PgOSC) gene family is known to play important roles in ginsenoside biosynthesis, but few members of the gene family have been functionally studied.MethodsThe PgOSC gene family has been studied by an integrated analysis of gene expression-ginsenoside content correlation, gene mutation-ginsenoside content association and gene co-expression network, followed by functional analysis through gene regulation.ResultsWe found that five of the genes in the PgOSC gene family, including two published ginsenoside biosynthesis genes and three new genes, were involved in ginsenoside biosynthesis. Not only were the expressions of these genes significantly correlated with ginsenoside contents, but also their nucleotide mutations significantly influenced ginsenoside contents. These results were further verified by regulation analysis of the genes by methyl jasmonate (MeJA) in ginseng hairy roots. Four of these five PgOSC genes were mapped to the ginsenoside biosynthesis pathway. These PgOSC genes expressed differently across tissues, but relatively consistent across developmental stages. These PgOSC genes formed a single co-expression network with those published ginsenoside biosynthesis genes, further confirming their roles in ginsenoside biosynthesis. When the network varied, ginsenoside biosynthesis was significantly influenced, thus revealing the molecular mechanism of ginsenoside biosynthesis.ConclusionAt least five of the PgOSC genes, including the three newly identified and two published PgOSC genes, are involved in ginsenoside biosynthesis. These results provide gene resources and knowledge essential for enhanced research and applications of ginsenoside biosynthesis in ginseng.     

Methyl jasmonate induces ATP biosynthesis deficiency and accumulation of proteins related to secondary metabolism in Catharanthus roseus (L.) G. hairy roots

Jasmonates are specific signal molecules in plants that are involved in a diverse set of physiological and developmental processes. However, methyl jasmonate (MeJA) has been shown to have a negative effect on root growth and, so far, the biochemical mechanism for this is unknown. Using Catharanthus roseus hairy roots, we were able to observe the effect of MeJA on growth inhibition, cell disorganization and cell death of the root cap. Hairy roots treated with MeJA induced the perturbation of mitochondrial membrane integrity and a diminution in ATP biosynthesis. Furthermore, several proteins were identified that were involved in energy and secondary metabolism; the changes in accumulation of these proteins were observed with 100 μM MeJA. In conclusion, our results suggest that a switch of the metabolic fate of hairy roots in response to MeJA could cause an increase in the accumulation of secondary metabolites. This is likely to have important consequences in the production of specific alkaloids important for the pharmaceutical industry.     

Gene ontology study of methyl jasmonate-treated and non-treated hairy roots of <Emphasis Type="Italic">Panax ginseng</Emphasis> to identify genes involved in secondary metabolic pathway

The roots of Panax ginseng C.A. Meyer, known as Korean ginseng have been a valuable and important folk medicine in East Asian countries. It mainly used to maintain the homeostasis of the human body, with the presence of ginsenosides and non-saponin compounds like phenol compounds, acidic polysaccharides and polyethylene compounds. Functional genomics aid to annotate EST sequences based on gene ontology. In this study, we focused, genes which involve in secondary metabolic pathways and to visualize temporal changes of gene expression in ginseng hairy roots with methyl ester methyl jasmonate (MeJA) along with non-treated hairy roots. A 5.774 EST clones were clustered and assembled into 501 contigs and 2.955 singletons. Annotations categorized with molecular functions, biological processes, cellular compounds of gene ontological terms and biochemical functions, enzyme commission number, and metabolic pathways are assigned through Kyoto Encyclopedia of Genes and Genomes database. Comparatively, EST sequences are assigned to cellular process, metabolic process, biotic and abiotic stress stimuli, developmental and biological regulations and transports are up-regulated 2–3 fold in MeJA treated hairy roots. 46 different sub groups of enzymes found in the MeJA treated plants. These annotated ESTs represents a significant proportion of the P. ginseng and provides molecular resource for develop microarray to study genes expressions to development, metabolism and reproduction.     

Characterization of Squalene synthase Gene from Chlorophytum borivilianum (Sant. and Fernand.)

Saponins are important group of secondary metabolites known for their pharmacological properties. Chlorophytum borivilianum contains high amount of saponins and is thus, recognized as an important medicinal plant with aphrodisiac properties. Though the plant is well known for its pharmaceutical properties, there is meager information available about the genes and enzymes responsible for biosynthesis of saponins from this plant. Squalene synthase (SqS) is the key enzyme of saponin biosynthesis pathway and here, we report cloning and characterization of SqS gene from C. borivilianum. A full-length CbSqS cDNA consisting of 1,760 bp was cloned which contained an open reading frame (ORF) of 1,233 bp, encoding a protein of 411 amino acids. Analysis of deduced amino acid sequence of CbSqS predicted the presence of conserved isoprenoid family domain and catalytic sites. Phylogenetic analysis revealed that CbSqS is closer to Glycine max and monocotyledonous plants. 3D structure prediction using various programs showed CbSqS structure to be similar to SqS from other species. C-terminus truncated recombinant squalene synthase (TruncCbSqS) was expressed in E. coli M15 cells with optimum expression induced with 1 mM IPTG at 37 °C. The gene expression level was analyzed through semi-quantitative RT-PCR and was found to be higher in leaves as compared to the roots.     

Regulation and differential expression of protopanaxadiol synthase in Asian and American ginseng ginsenoside biosynthesis by RNA interferences

Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolium), are thought to be representative plant of Panax species, have important commercial value and are used in worldwide. Panax species produces triterpene saponins called ginsenosides, which are classified into two groups by the skeleton of aglycones, namely dammarane-type and oleanane-type. Dammarane-type ginsenosides dominate over oleanane-type not only in amount but also in structural varieties. Researches shows that the saponins content in American ginseng is higher than that in Asian ginseng, the higher part of ginsenosides is from dammarane-type biosynthesis. It has been proposed that protopanaxadiol derived from dammarenediol-II, is a key hydroxylation by cytochrome P450 for the biosynthesis of ginsenosides, and the gene number of protopanaxadiol synthase has been published independent in Asian ginseng (PgCYP716A47). However, little is known about genes involved in hydroxylation and glycosylation in American ginseng ginsenoside biosynthesis. Here, we first cloned and identified a P450 gene named PqD12H encoding enzymes catalyzed dammarenediol-II to protopanaxadiol by RT-PCR using degenerate primers designed based on sequence homology. In vitro, the ectopic expression of PqD12H in recombinant WAT21 yeast resulted in protopanaxadiol production after dammarenediol-II was added to the culture medium. In vivo, we established both PgCYP716A47 and PqD12H RNAi transgenic. The RT-PCR and HPLC analysis of the final products of protopanaxadiol and protopanaxatriol showed a result that declined level of protopanaxadiol-type and protopanaxatriol-type ginsenosides. It suggested that the P450 synthase content or expression in American ginseng exceed than in Asian ginseng. The result elucidated the evolution relationship of P450s and the reason of different saponins content among Panax species.     

Fungal sensitivity to and enzymatic deglycosylation of ginsenosides

A ginseng pathogen, Cylindrocarpon destructans, and five nonpathogens were tested for their sensitivity to a total ginsenoside fraction (T-GF), a protopanaxadiol-type ginsenoside fraction (PPD-GF) and a protopanaxatriol-type ginsenoside fraction (PPT-GF) from the roots of Panax ginseng C.A. Meyer. The results showed that T-GF inhibited growth of the five ginseng nonpathogens, while it promoted growth of the ginseng pathogen C. destructans. PPT-GF and PPD-GF both inhibited the growth of the five ginseng nonpathogens, although the activity of PPT-GF was higher than that of PPD-GF. PPT-GF and PPD-GF exhibited different activities on C. destructans: PPT-GF inhibited its growth, whereas PPD-GF significantly enhanced its growth. The subsequent analysis of enzymatic degradation of ginsenosides by the test fungi showed that C. destructans can consecutively hydrolyze the terminal monosaccharide units from the sugar chains attached at C3 and C20 in PPD-type ginsenosides by extracellular glycosidase activity to yield four major products, gypenoside XVII (G-XVII), compound O, compound Mb and the ginsenoside F₂. By contrast, the ginseng nonpathogens Aspergillus nidulans and Cladosporium fulvum have no extracellular glycosidase activity toward sugar chains attached to C3 in PPD-type ginsenosides. These results indicated that ginsenosides might act as host chemical defenses, while the ginseng root pathogenic fungi might counter their toxicity by converting PPD-type ginsenosides into growth or host recognition factors. The ability of ginseng root pathogens to deglycosylate PPD-type ginsenosides may be a pathogenicity factor.     

Influence of salicylic acid and methyl jasmonate elicitation on anthocyanin production in callus cultures of Rosa hybrida L.

This study was undertaken to investigate the effects of salicylic acid (SA) and methyl jasmonate (MeJA) on anthocyanin induction, biomass accumulation, and color value (CV) indices for both pigment content (PC) and pigment production (PP) in callus cultures of Rosa hybrida cv. Pusa Ajay. A concentration-dependent response was exhibited by cultures on SA and MeJA at different concentrations individually or in combinations to Euphorbia millii medium supplemented with 204.5 mM sucrose, 2.45 μM indole butyric acid and 2.33 μM kinetin. There was positive influence on both callus biomass and anthocyanin accumulation. Treatment with 0.5 μM MeJA was most effective in inducing anthocyanin biosynthesis in callus cultures. Anthocyanin accumulation in callus cultures was enhanced with the addition of SA and MeJA, but these did not differ significantly from control for the number of days required for pigment initiation and for color intensification. Moreover, the addition of 0.5 μM MeJA alone resulted in a higher frequency of color response (97.25 %), PC (3.48 ± 0.07 CV g^?1 FW), and PP (1.56 ± 0.03 CV test tube^?1) over control. In contrast, the presence of higher levels of SA (400 μM) and MeJA (5.0 μM) reduced frequency of color response, as well as levels of PC and PP. MeJA did not increase biomass accumulation but promoted frequency of color response, PC and PP. Hence, it was suggested that 0.5 μM MeJA promoted anthocyanin production in rose callus cultures. Significant correlation was found between frequency of response and each of the PC (r = 0.988) and PP (r = 0.990). Furthermore, PC and PP were also highly correlated (r = 0.998).     

Extraction of ginsenosides from fresh ginseng roots (Panax ginseng C.A. Meyer) using commercial enzymes and high hydrostatic pressure

A combination of high hydrostatic pressure (HHP) and enzymatic hydrolysis (HHP-EH) was applied for the extraction of ginsenosides from fresh ginseng roots (Panax ginseng C.A. Myer). The highest yield of ginsenosides was obtained by using a mixture of three enzymes (Celluclast + Termamyl + Viscozyme) along with HHP (100 MPa, at 50 °C for 12 h) in comparison to control samples (no enzymes, atmosphere pressure, P < 0.05). Total ginsenosides increased by 184 % while Rg1 + Rb1 increased by 273 %. Application of these conditions significantly increased total ginsenosides by 49 % and Rg1 + Rb1 by 103 % compared to HHP treatment alone (P < 0.05). The effect of HHP on increased yield of ginsenosides is likely due in part, to acceleration of enzyme activity. Thus HHP-EH significantly improves the extraction of ginsenosides from fresh ginseng roots.     

Development of interspecies hybrids to increase ginseng biomass and ginsenoside yield

Key message

Interspecific hybrids between Panax ginseng and P. quinquefolius results in hybrid vigor and higher ginsenoside contents.

Abstract

Ginseng is one of the most important herbs with valued pharmaceutical effects contributing mainly by the presence of bioactive ginsenosides in the roots. However, ginseng industry is impeded largely by its biological properties, because ginseng plants are slow-growing perennial herbs with lower yield. To increase the ginseng yield and amounts of ginsenosides, we developed an effective ginseng production system using the F₁ progenies obtained from the interspecific reciprocal cross between two Panax species: P. ginseng and P. quinquefolius. Although hybrid plants show reduced male fertility, F₁ hybrids with the maternal origin either from P. ginseng or P. quinquefolius displayed heterosis; they had larger roots and higher contents of ginsenosides as compared with non-hybrid parental lines. Remarkably, the F₁ hybrids with the maternal origin of P. quinquefolius had much higher ginsenoside contents, especially ginsenoside Re and Rb1, than those with the maternal origin of P. ginseng. Additionally, non-targeted metabolomic profiling revealed a clear increase of a large number of primary and secondary metabolites including fatty acids, amino acids and ginsenosides in hybrid plants. To effectively identify the F₁ hybrids for the large-scale cultivation, we successfully developed a molecular marker detection system for discriminating F₁ reciprocal hybrids. In summary, this work provided a practical system for reciprocal hybrid ginseng production, which would facilitate the ginseng production in the future.